An organ and a tissue damaged by an injury, a disease or aging require promotion of regeneration to recover its function. Particularly, a substantial organ such as heart, liver, kidney, or pancreas is essential for maintaining life, and thus hypofunction and a function thereof causes a death. Therefore transplantation treatment such as organ transplantation is popularly conducted intending life saving. However, the number of donor is generally insufficient, and thus a new approach to solve the problem is necessary.
In recent years, regeneration medicine by using a stem cell, which is present in an embryo or an adult and may have an ability of dividing indefinitely to differentiate in one or a plurality of directions, to prepare the tissue and the organ for repairing a defect tissue, attracts attention as a therapeutic method without the defect of the conventional organ transplantation.
Specifically, it is exemplified that following propagation, the stem cell is grown and then differentiated to be used for cell transplantation and, through use of an artificial support tissue and artificial tissue construction, the artificial tissue is transplanted into a living body and is used as an artificial organ. It is expected that using the stem cell for cell transplantation therapy and tissue engineering allows solving the problem, which includes a defect of a tissue after a transplanting piece is resected from the donor and the shortage of the donor, in transplantation therapy including conventional autotransplantation.
The stem cell has been identified from many organs such as a blood vessel, nerve, blood, cartilage, bone, liver, pancreas. Particularly among them, a totipotent stem cell having the ability of differentiating in all cell types gets a lot of attention as a cell that can provide easily a cell and a tissue used for the aforementioned regeneration medicine field and also drug creation and gene therapy.
Known examples of the totipotent stem cell are Embryonic Stem (hereafter ES) cell and Embryonic Germ (hereafter EG) cell. The ES cell is a cell line isolated from an Inner Cell Mass (ICM) of a blastocyst stage of a mouse (Evans et al., Nature, 292: 154, 1981). Cells consisting an individual are derived from a primary ectoderm produced from an Inner Cell Mass (hereafter ICM) of the blastocyst stage or an epiblast of a gastrula, and the ICM and the epiblast are a stem cell group having the totipotency. The ES cell has the ability of the differentiation into various kinds of tissues to form the individual and forms a normal embryo and a chimeric embryo to differ into any mature cells of the adult. In addition, the ES cell has the ability of producing such various cells as blood cells, myocardial cell, vascular endothelial cell, nerve cell, pigment cell, pancreatic incretion cell depending on the condition of in vitro differentiation induction (Nakano, T. Saisin Igaku Bessatsu Regeneration medicine: 81-89. 2000).
The EG cell is a cell line established by culturing a primordial germ cell in presence of LIF (Leukemia Inhibitory Factor) and bFGF (basic Fibroblast Growth Factor) (Matsui et al., Cell 70: 84. 1992; Resnic et al., Nature 359: 550. 1992) and has ability of differentistion into various tissues as in the case of the ES cell.
In recent years, the ES cell lines established in animals other than the mouse were reported, and it was shown that they have multidifferentiation ability similar to that of the mouse ES cell (bovine ES cell: Schellander et al., Theriogenology, 31: p15-17, 1989, swine ES cell: Strojek et al., Theriogenology 33: p901, 1990, sheep ES cell: primate Handyside, Roux's Arch. De v. Biol., 196: p185, 1987, hamster ES cell: Doetschman et al., Dev. Biol., 127: p224, 1988, Rhesus macaque ES cell: Thomson et al., Proc. Natl. Acad. Sci. USA, 92: p7844, 1995, marmoset ES cell: Thomson et al., Biology of Production 55: p254, 1996, human ES cell: Thomson et al., Science, 282: p1145, 1998, Reubinoff et al., Nature Biotech 18: p399, 2000, cynomolgus monkey ES cell: Suemori et al., Dev. Dyn. 222: p273, 2001).
In order to maintain undifferentiation of the ES cell, it is generally necessary to co-culture a fibroblast originated from a mouse fetus as a feeder cell. The same method is used for maintaining undifferentiation of the ES cell line of a primate (Thomson et al., Proc. Natl. Acad. Sci. USA 92: p7844, 1995, Thomson et al., Science: 282: p1145, 1998, Reubinoff et al., Nature Biotech 18: p399, 2000).
However, preparation of the mouse primary fibroblast is complicated as follows. A 13.5 to 15.5-day old embryo is taken out from a pregnant mouse, the embryo is degraded by enzyme treatment, and the fibroblast yielded is collected on a dish. Since this cell is primary cell, quality management is complicated, management on a GMP-matched level is difficult, and the ability of maintaining undifferentiation may be different depending on the embryo used. As an ES cell culture method without this complicated preparation work, there is a method of using an STO cell (ATCC 56-X) which is a cell line of the fibroblast of a mouse embryo. However, the STO cell's ability of maintaining undifferentiation of the ES cell is variable, and thus, for stable culture of the ES cell, the mouse primary fibroblast is superior.
In recent years, an infection case of an endogenous virus across different animal species was reported (van der Laan et al., Nature 407: p90, 2000). The culture method aiming use of the human ES cell for medicine requires developing the culture method without contact between cells of different animal species as possible. Therefore, the above culture method for maintaining undifferentiation of the ES cell by using cells derived from the mouse is not suitable for culturing the ES cell aiming to use for medicine.
As the method for culturing a primate ES cell using no mouse-derived feeder cell, a method of adding a component secreted by the mouse primary fibroblast to a culture medium has been reported (for example, Japanese Patent Laid-open Publication No. 2001-17163). However, also in this case, the ES cell under culturing is exposed to an unidentified factor secreted from the mouse cell, and therefore the ES cell cultured in such an environment is not suitable for the use in medicine. Moreover, a danger of infection of the endogenous virus remains Consequently, the defect caused by coculture with the mouse primary fibroblast is not solved at all.
As the method for culture for maintaining undifferentiation of the mouse ES cell without using the mouse-derived feeder cell and the component secreted by the mouse feeder cell, a culture method of using a culture dish coated with gelatin has been already known. However, in this method, leukemia inhibitory factor (LIF) must be added to the culture medium (for example, Smith et al., Dev. Biol. 121: p1, 1987). LIF is a cytokine and therefore, has problems of high cost and a bad preservation performance, resulting in unsuitableness for mass culture. In addition, an effect of LIF is limited to the ES cell derived from a very specific mouse line (129/sv line and C57BL/6 line) and shows no distinct effect on animals of other species. Especially, for the primate ES cell, it has been known that addition of LIF to the culture medium alone does not allow keeping the undiffentiation status (for example, Thomson et al., Proc. Natl. Acad. Sci. USA 92: p7844, 1995; and Thomson et al., Science 282: p1145, 1998).
On the other hand, as a low molecular weight compound which amplifies an LIF's action of maintaining undifferentiation of an embryonic stem cell, PD98059 (Cell Signaling Technology Corp. made) is reported. However, the action of PD98059 depends on LIF and does not express independently the effect (Burdon et al., Dev. Biol. 210: p30, 1999) and, hence, the aforementioned problem is not solved.
Consequently, so far, there was no differentiation inhibiting agent enabling safe mass culture of the totipotent stem cell at a low cost, and no method of safe mass culture of the totipotent stem cell at the low cost has been known. The differentiation inhibiting agent according to the present invention comprises a low molecular weight compound as the active ingredient. It has been not so far known that a low molecular weight compound maintains the undifferentiated status of the totipotent stem cell. Therefore, the action of maintaining undifferentiation of the totipotent stem cell which is possessed by the low molecular weight compound represented by the formulae (1) to (10) shown in this specification, has not been known at all so far.